Designed metalloprotein stabilizes a semiquinone radical

Area of Science:

• Biochemistry
• Protein Engineering
• Organic Chemistry

Background:

• Enzymes utilize binding energy to stabilize high-energy substrate states.
• Reactive organic radicals are often unstable in aqueous environments.

Purpose of the Study:

• To design a de novo metalloprotein capable of stabilizing a reactive organic radical.
• To investigate the stabilization mechanism of the radical semiquinone form of 3,5-di-tert-butylcatechol.

Main Methods:

• De novo protein design and synthesis.
• Solution Nuclear Magnetic Resonance (NMR) spectroscopy.
• Molecular dynamics (MD) simulations.
• Spectrochemical redox titrations.

Main Results:

• The designed Zn(II) metalloprotein tightly binds and stabilizes the semiquinone radical.
• Substrate stabilization occurs via metal-ligand interactions and hydrophobic group burial in the active site.
• The protein reduced the electrochemical midpoint potential for semiquinone formation by approximately 400 mV (9 kcal mol(-1)).

Conclusions:

• Harnessing binding energy to a designed metalloprotein can drastically alter the inherent chemical properties of a radical.
• This work provides a foundation for creating engineered enzymes with radical cofactors to perform challenging chemical transformations.